General co-expression vectors for the overexpression of heterodimeric protein complexes in Escherichia coli
General co-expression vectors for the overexpression of heterodimeric protein complexes in Escherichia coli
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DOI：
10.1016/j.pep.2004.07.016
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发表时间：
2004-11-01
影响因子：
1.6
通讯作者：
Hao, W
中科院分区：
文献类型：
--
作者：
Dzivenu, OK;Park, HH;Hao, W
我们已经设计并构建了一对新的细菌共表达载体，以促进生产大量的重组多蛋白复合物的生化，生物物理和结构研究。pOKD 4（卡那霉素抗性）和pOKD 5（氨苄青霉素抗性）是pACYC 177克隆和pET 26 b表达载体的衍生物。结果，pOKD 4和pOKD 5是含有p15 A复制起点的基于T7的表达质粒。该特征允许pOKD 4或pOKD 5与大多数大肠杆菌表达载体（包括流行的pET表达载体）共存于同一细菌细胞中。pOKD 4和pOKD 5载体已被改造为具有与pET 26 b完全相同的多克隆位点，从而允许相对容易地来回穿梭基因。这对新型共表达载体的有效性和通用性已成功应用于在大肠杆菌中生产大量活性DFF 40/DFF 45异二聚体蛋白复合物。大肠杆菌进行详细的生化和结构研究。(C)2004年爱思唯尔公司All rights reserved.
We have designed and constructed a novel pair of bacterial co-expression vectors to facilitate the production of substantial amounts of recombinant multiprotein complexes for biochemical, biophysical, and structural studies. pOKD4 (kanamycin-resistant) and pOKD5 (ampicillin-resistant) are derivatives of pACYC177 cloning and pET26b expression vectors. As a result, pOKD4 and pOKD5 are T7-based expression plasmids containing the p15A origin of replication. This feature permits either pOKD4 or pOKD5 to co-exist in the same bacterial cell with most Escherichia coli expression vectors including the popular pET expression vectors. The pOKD4 and pOKD5 vectors have been engineered to possess exactly the same multiple cloning sites as pET26b thus allowing for the relatively easy shuttling of genes to and fro. The efficacy and versatility of this novel pair of co-expression vectors was successfully applied to the production of significant amounts of active DFF40/DFF45 heterodimeric protein complex in E. coli for detailed biochemical and structural studies. (C) 2004 Elsevier Inc. All rights reserved.